Monofunctional glycosyltransferase of Staphylococcus aureus

ABSTRACT

The invention provides isolated nucleic acid compounds encoding a novel MTG of Staphylococcus aureus. Also provided are vectors and transformed heterologous host cells for expressing the MTG and a method for identifying compounds that bind and/or inhibit the enzymatic activity of the MTG.

This application is a divisional of U.S. application Ser. No.08/771,716, filed Dec. 20, 1996, now U.S. Pat. No. 5,922,540, thecontents of which are herein incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

This invention relates to recombinant DNA technology. In particular theinvention pertains to the cloning of a gene, mtg, encoding a novelmonofunctional glycosyltransferase, from Staphylococcus aureus and theuse of said gene and its encoded protein in a screen for new inhibitorsof bacterial cell wall biosynthesis.

The emergence of antibiotic resistance in common pathogenic bacterialspecies has justifiably alarmed the medical and research communities.The emergence and rapid spread of beta-lactam resistance has beenparticularly problematic. Increasingly, the only drug that can be usedto treat infections with MDR (multiple drug resistant) organisms isvancomycin, and there is considerable concern that the bacteria couldalso develop resistance to vancomycin.

The bacterial cell wall comprises a peptidoglycan layer which providesmechanical rigidity for the bacterium. The peptidoglycan layer iscomposed of a sugar backbone (alternating residues ofN-acetylglucosamine and N-acetylmuramic acid are polymerized through atransglycosylation reaction) attached to a pentapeptide (also referredto as "stem peptide") containing D and L amino acid residues. Adjacentstem peptide residues are covalently crosslinked during maturation ofthe peptidoglycan.

During formation of the mature peptidoglycan, a lipid-linkeddisaccharide-pentapeptide is translocated across the cytoplasmicmembrane, exposing the pentapeptide sidechains to the cell surface. Thefully mature peptidoglycan structure is obtained followingtransglycosylation and transpeptidation enzymatic reactions. Severalenzymes appear to be involved in the transglycosylation andtranspeptidation polymerizaion reactions, most notably the bifunctionalhigh molecular weight PBPs. Interestingly, transglycosylation activityis also found in monofunctional enzymes known as monofunctionalglycosyltransferases (MTG's). The MTG is a target for the development ofnew inhibitors of cell wall synthesis.

SUMMARY OF THE INVENTION

The present invention provides, inter alia, isolated nucleic acidmolecules that encode an MTG from Staphylococcus aureus. The inventionalso provides protein products encoded by the gene, in substantiallypurified form.

Having the cloned mtg gene of Staphylococcus aureus enables theproduction of recombinant MTG protein and derivatives thereof for theimplementation of assays and screens to identify new inhibitorycompounds targeted at the peptidoglycan biosynthetic pathway.

In one embodiment the present invention relates to isolated gene mtgthat encodes novel Staphylococcus aureus MTG, said gene comprising thenucleotide sequence identified as SEQ ID NO. 1.

In another embodiment the present invention relates to a novel proteinmolecule, MTG, wherein said protein molecule comprises the sequenceidentified as SEQ ID NO. 2.

In another embodiment, the present invention relates to a soluble formof MTG (designated MTG^(S)) wherein MTG^(S) comprises amino acidresidues 68 through 269 of SEQ ID NO.2.

In a further embodiment the present invention relates to a ribonucleicacid molecule encoding MTG protein, said ribonucleic acid moleculecomprising the sequence identified as SEQ ID NO. 3 or fragment thereof.

In yet another embodiment, the present invention relates to arecombinant DNA vector that incorporates the Staphylococcus aureus mtggene in operable linkage to gene expression sequences enabling said mtggene to be transcribed and translated in a host cell.

In still another embodiment the present invention relates to homologousor heterologous host cells that have been transformed or transfectedwith a vector carrying the cloned mtg gene from Staphylococcus aureussuch that said gene is expressed in the host cell.

In a still further embodiment, the present invention relates to a methodfor identifying compounds that bind and/or inhibit the enzymaticactivity of the Staphylococcus aureus MTG protein or fragment thereof.

DESCRIPTION OF THE DRAWING

FIGURE. Plasmid pPSR-23, useful for high level expression of theStaphylococcus aureus mtg^(S) gene of the present invention in theheterologous procaryotic host cell Eschericia coli.

DEFINITIONS

"mtg" refers to the Staphylococcus aureus genomic DNA sequence encodingMTG and fragments thereof.

"mtg^(S) " refers to a portion of mtg that encodes MTG^(S) comprisingnucleotide residues 202 through 807 of SEQ ID NO.1.

"MTG" as used herein may refer to the native monofunctionalglycosyltransferase or to a portion thereof.

The terms "cleavage" or "restriction" of DNA refers to the catalyticcleavage of the DNA with a restriction enzyme that acts only at certainsequences in the DNA (viz. sequence-specific endonucleases). The variousrestriction enzymes used herein are commercially available and theirreaction conditions, cofactors, and other requirements are used in themanner well known to one of ordinary skill in the art. Appropriatebuffers and substrate amounts for particular restriction enzymes arespecified by the manufacturer or can readily be found in the literature.

The term "fusion protein" denotes a hybrid protein molecule not found innature comprising a translational fusion or enzymatic fusion in whichtwo or more different proteins or fragments thereof are covalentlylinked on a single polypeptide chain.

"Functional domain" refers to a region of a protein having one or moredistinct biological functions, for example, enzymatic activity,transmembrane anchoring, DNA binding, etc. A functional domain comprisesa sequence of amino acids, the length of which and the identity of aminoacid residues therein, may or may not be critical to said function.

The term "plasmid" refers to an extrachromosomal genetic element. Thestarting plasmids herein are either commercially available, publiclyavailable on an unrestricted basis, or can be constructed from availableplasmids in accordance with published procedures. In addition,equivalent plasmids to those described are known in the art and will beapparent to the ordinarily skilled artisan.

"Recombinant DNA cloning vector" as used herein refers to anyautonomously replicating agent, including, but not limited to, plasmidsand phages, comprising a DNA molecule to which one or more additionalDNA segments can or have been added.

The term "recombinant DNA expression vector" as used herein refers toany recombinant DNA cloning vector, for example a plasmid or phage, inwhich a promoter and other regulatory elements are present to enabletranscription of the inserted DNA.

The term "vector" as used herein refers to a nucleic acid compound usedfor introducing exogenous DNA into host cells. A vector comprises anucleotide sequence which may encode one or more protein molecules.Plasmids, cosmids, viruses, and bacteriophages, in the natural state orwhich have undergone recombinant engineering, are examples of commonlyused vectors.

The terms "complementary" or "complementarity" as used herein refers tothe capacity of purine and pyrimidine nucleotides to associate throughhydrogen bonding in double stranded nucleic acid molecules. Thefollowing base pairs are complementary: guanine and cytosine; adenineand thymine; and adenine and uracil.

"Isolated nucleic acid compound" refers to any RNA or DNA sequence,however constructed or synthesized, which is locationally distinct fromits natural location.

A "primer" is a nucleic acid fragment which functions as an initiatingsubstrate for enzymatic or synthetic elongation of, for example, anucleic acid molecule.

The term "promoter" refers to a DNA sequence which directs transcriptionof DNA to RNA.

A "probe" as used herein is a labeled single-stranded nucleic acidcompound of greater than 5 nucleotide residues that has the potential tohybridize with another nucleic acid compound.

The term "hybridization" as used herein refers to the phenomenon bywhich a single-stranded nucleic acid molecule joins with a complementarysingle-stranded nucleic acid molecule through nucleotide base pairing."Selective hybridization" refers to hybridization that occurs underconditions of high stringency. The extent of hybridization depends upona number of variable and constant parameters, for example, the degree ofcomplementarity, the stringency of hybridization, and the length ofhybridizing strands.

The term "stringency" refers to hybridization conditions. Highstringency conditions disfavor non-homologous basepairing. Lowstringency conditions have the opposite effect. Stringency may bealtered, for example, by changing the temperature and saltconcentration.

"Transglycosylation" refers to an enzymatic reaction in which the sugarresidues of lipid-linked disaccharide pentapeptide molecules arepolymerized during the formation of the peptidoglycan structure of thebacterial cell wall.

DETAILED DESCRIPTION

The mtg gene (SEQ ID NO.1) of the present invention encodes a novel MTGenzyme of Staphylococcus aureus (SEQ ID NO. 2). The mtg gene disclosedherein comprises a DNA sequence of 807 nucleotide base pairs (SEQ ID NO.1). There are no intervening sequences. Those skilled in the art willrecognize that owing to the degeneracy of the genetic code (i.e. 64codons which encode 20 amino acids), numerous "silent" substitutions ofnucleotide base pairs could be introduced into the sequence identifiedas SEQ ID NO. 1 without altering the identity of the encoded aminoacid(s) or protein product. All such substitutions are intended to bewithin the scope of the invention.

The MTG protein defined by SEQ ID NO.2 comprises a membrane-boundprotein of 269 amino acid residues. The MTG protein of the presentinvention may be modified by deletion of amino acid residues 1 through67 at the amino terminal end. Deletion of this region results in removalof the transmembrane region and the production of a soluble form,MTG^(S) which retains the transglycosylase domain of the native enzyme.Another modified form of MTG, which retains the transmembrane region,comprises amino acid residues 17 through 269 of SEQ ID NO.2.

Gene Isolation Procedures

Those skilled in the art will recogize that the gene of the presentinvention may be obtained by a plurality of applicable genetic andrecombinant DNA techniques including, for example, polymerase chainreaction (PCR) amplification, or de novo DNA synthesis. (See e.g., J.Sambrook et al. Molecular Cloning, 2d Ed. Chap. 14 (1989)).

Methods for constructing gene libraries in a suitable vector such as aplasmid or phage for propagation in procaryotic or eucaryotic cells arewell known to those skilled in the art. [See e.g. J. Sambrook et al.Supra]. Suitable cloning vectors are widely available.

Skilled artisans will recognize that the mtg gene of Staphylococcusaureus comprising the present invention or fragment thereof could beisolated by PCR amplification of Staphylococcus aureus genomic DNA orcDNA using oligonucleotide primers targeted to any suitable region ofSEQ ID NO. 1. Methods for PCR amplification are widely known in the art.See e.g. PCR Protocols: A Guide to Method and Application, Ed. M. Inniset al., Academic Press (1990). The amplification reaction comprisesgenomic DNA, suitable enzymes, for example Taq DNA polymerase, primers,and buffers, and is conveniently carried out in a DNA Thermal Cycler(Perkin Elmer Cetus, Norwalk, Conn.). A positive result is determined bydetecting an appropriately-sized DNA fragment following agarose gelelectrophoresis.

Protein Production Methods

One embodiment of the present invention relates to the substantiallypurified MTG protein or fragment thereof, for example MTG^(S).

Skilled artisans will recognize that the protein of the presentinvention can be synthesized by any number of different methods. Theamino acid compounds of the invention can be made by chemical methodswell known in the art, including solid phase peptide synthesis orrecombinant methods. Both methods are described in U.S. Pat. No.4,617,149, incorporated herein by reference.

The principles of solid phase chemical synthesis of polypeptides arewell known in the art and may be found in general texts in the area.See, e.g., H. Dugas and C. Penney, Bioorganic Chemistry (1981)Springer-Verlag, New York, 54-92. For example, peptides may besynthesized by solid-phase methodology utilizing an Applied Biosystems430A peptide synthesizer (Applied Biosystems, Foster City, Calif.) andsynthesis cycles supplied by Applied Biosystems. Protected amino acids,such as t-butoxycarbonyl-protected amino acids, and other reagents arecommercially available from many chemical supply houses.

The proteins of the present invention can also be produced byrecombinant DNA methods using the cloned mtg gene or fragment thereof,as disclosed herein. Recombinant methods are preferred if a high yieldof protein is desired. Expression of said cloned gene can be carried outin a variety of suitable host cells well known to those skilled in theart. In a recombinant method the mtg or mtg^(S) gene or variants thereofare introduced into a host cell by any suitable means, well known tothose skilled in the art. While chromosomal integration of a cloned mtggene or variant thereof is within the scope of the present invention, itis preferred that the gene be cloned into a suitable extrachromosomallymaintained expression vector so that the coding region of the gene isoperably linked to a constitutive or inducible promoter.

The basic steps in the recombinant production of an MTG or MTG^(S) ofthe present invention are:

a) constructing a natural, synthetic or semi-synthetic DNA encoding saidMTG or MTG^(S) protein;

b) integrating said DNA into an expression vector in a manner suitablefor expressing said MTG or MTG^(S) ;

c) transforming or otherwise introducing said vector into an appropriateeucaryotic or procaryotic host cell forming a recombinant host cell,

d) culturing said recombinant host cell in a manner enabling expressionof said protein; and

e) recovering and substantially purifying said protein by any suitablemeans, well known to those skilled in the art.

Expressing Recombinant MTG Proteins in Procaryotic and Eucaryotic HostCells

In general, procaryotes are used for cloning DNA sequences and forconstructing the vectors of the present invention. Procaryotes may alsobe employed in the production of the MTG proteins of the presentinvention. For example, the Escherichia coli K12 strain 294 (ATCC No.31446) is particularly useful for the procaryotic expression of foreignproteins. Other strains of E. coli, bacilli such as Bacillus subtilis,enterobacteriaceae such as Salmonella typhimurium or Serratiamarcescans, various Pseudomonas species and other bacteria, such asStreptomyces, may also be employed as host cells in the cloning andexpression of the recombinant proteins of this invention.

Promoter sequences suitable for driving the expression of genes inprocaryotes include β-lactamase [e.g. vector pGX2907, ATCC 39344,contains a replicon and β-lactamase gene], lactose systems [Chang etal., Nature (London), 275:615 (1978); Goeddel et al., Nature (London),281:544 (1979)], alkaline phosphatase, and the tryptophan (trp) promotersystem [vector pATH1 (ATCC 37695) which is designed to facilitateexpression of an open reading frame as a trpE fusion protein under thecontrol of the trp promoter]. Hybrid promoters such as the tac promoter(isolatable from plasmid pDR540, ATCC-37282) are also suitable. Stillother promoters, such as that from bacteriophage T7, whose nucleotidesequences are generally known, enable one of skill in the art to ligatesuch promoter sequences to DNA encoding the proteins of the instantinvention using linkers or adapters to supply any required restrictionsites. Still other promoters are useful for gene expression in S.pneumoniae, for example the ami promoter (J. P. Claverys et al."Construction and evaluation of new drug-resistance cassettes for genedisruption mutagenesis in Staphylococcus aureus, using an ami testplatform," Gene (1995) 123-128) Promoters for use in bacterial systemswill also contain a Shine-Dalgarno sequence operably linked to the DNAencoding the desired polypeptides. These examples are illustrativerather than limiting.

In addition to procaryotes, a variety of eucaryotic microorganisms suchas yeast are suitable host cells. The yeast Saccharomyces cerevisiae isthe most commonly used eucaryotic microorganism. A number of otheryeasts such as Kluyveromyces lactis are also suitable. For expression inSaccharomyces, the plasmid YRp7 (ATCC-40053), for example, may be used.See, e.g., L. Stinchcomb, et al., Nature, 282:39 (1979); J. Kingsman etal., Gene, 7:141 (1979); S. Tschemper et al., Gene, 10:157 (1980).Plasmid YRp7 contains the TRP1 gene which provides a selectable markerfor use in a trp1 auxotrophic mutant.

Purification of Recombinantly-Produced MTG and MTG^(S)

An expression vector carrying the cloned mtg or mtg^(S) ofStaphylococcus aureus or fragment thereof is transformed or transfectedinto a suitable host cell using standard methods. Cells that contain thevector are propagated under conditions suitable for expression of theencoded MTG. For example, if the gene is under the control of aninducible promoter, suitable growth conditions would incorporate anappropriate inducer. Recombinantly-produced MTG^(S) or MTG protein maybe purified from cellular extracts of transformed cells by any suitablemeans. Recombinantly-produced MTG that contains the N-terminal portionof the protein is expected to be localized in the host cell membrane. Assuch, recombinant MTGs may also be recoverable from cell extracts andcell membranes by any suitable means, well known to those skilled in theart.

In a preferred process for protein purification the gene encoding theMTG or MTG^(S) of the present invention is modified at the 5' end toincorporate several histidine residues at the amino terminal end of therespective protein molecules. The "histidine tag" method enables asimplified protein purification known as "immobilized metal ion affinitychromatography" (IMAC), essentially as described in U.S. Pat. No.4,569,794, which hereby is incorporated by reference. The IMAC methodenables rapid isolation of substantially pure protein starting from acrude cellular extract.

Other embodiments of the present invention comprise isolated nucleicacid sequences. As skilled artisans will recognize, owing to thedegeneracy of the genetic code the proteins of the invention can beencoded by a multitude of different nucleic acid sequences. Becausethese alternative nucleic acid sequences would encode the same aminoacid sequences, the present invention further comprises these alternatenucleic acid sequences.

Nucleic acid sequences that encode SEQ ID NO:2 or subregion therein maybe produced using synthetic methods. The synthesis of nucleic acids iswell known in the art. See, e.g., E. L. Brown, R. Belagaje, M. J. Ryan,and H. G. Khorana, Methods in Enzymology, 68:109-151 (1979). The DNAsegments corresponding to the mtg or related gene sequence mtg^(S) couldbe generated using a conventional DNA synthesizing apparatus, such asthe Applied Biosystems Model 380A or 380B DNA synthesizers (AppliedBiosystems, Inc., 850 Lincoln Center Drive, Foster City, Calif. 94404)which employ phosphoramidite chemistry. Alternatively, phosphotriesterchemistry may be employed to synthesize the nucleic acids of thisinvention. [See, e.g., M. J. Gait, ed., Oligonucleotide Synthesis, APractical Approach, (1984).]

In an alternative and preferred methodology, namely PCR, the DNAsequence comprising a portion or all of SEQ ID NO:1 can be generatedfrom Staphylococcus aureus genomic DNA using suitable oligonucleotideprimers complementary to SEQ ID NO:1 or region therein, essentially asdescribed in U.S. Pat. No. 4,889,818, which hereby is incorporated byreference. Suitable protocols for performing the PCR are widely knownand are disclosed in, for example, PCR Protocols: A Guide to Method andApplications, Ed. Michael A. Innis et al., Academic Press, Inc. (1990).

The ribonucleic acids of the present invention may be prepared using thepolynucleotide synthetic methods discussed supra, or they may beprepared enzymatically using RNA polymerase to transcribe a suitable DNAtemplate.

The most preferred systems for preparing the ribonucleic acids of thepresent invention employ the RNA polymerase from the bacteriophage T7 orthe bacteriophage SP6. These RNA polymerases are highly specific,requiring the insertion of bacteriophage-specific sequences at the 5'end of the template to be transcribed. See, J. Sambrook, et al., supra,at 18.82-18.84.

This invention also provides nucleic acids, RNA or DNA, that arecomplementary to SEQ ID NO:1 or SEQ ID NO:3.

The present invention also provides probes and primers useful for avariety of molecular biology techniques including, for example,hybridization screens of genomic or subgenomic libraries. A nucleic acidcompound comprising SEQ ID NO:1, SEQ ID NO:3 or a complementary sequencethereof, or a fragment thereof, and which is at least 18 base pairs inlength, and which will selectively hybridize to Staphylococcus aureusDNA or mRNA encoding the MTG or fragment thereof of the presentinvention, is provided. Preferably, the 18 or more nucleotide bases areDNA. These probes and primers can be prepared by enzymatic methods wellknown to those skilled in the art (See e.g. Sambrook et al. supra). In amost preferred embodiment these probes and primers are synthesized usingchemical means as described above.

Another aspect of the present invention relates to recombinant DNAcloning vectors and expression vectors comprising the nucleic acids ofthe present invention. Many of the vectors encompassed within thisinvention are described above. The preferred nucleic acid vectors arethose that comprise DNA. The most preferred recombinant DNA vectorscomprise the isolated DNA sequence, SEQ ID NO:1. Plasmid pPSR-23 is anespecially preferred DNA vector for expressing the soluble form of theMTG of this invention in E. coli.

The skilled artisan understands that choosing the most appropriatecloning vector or expression vector depends upon a number of factorsincluding the availability of restriction enzyme sites, the type of hostcell into which the vector is to be transfected or transformed, thepurpose of the transfection or transformation (e.g., stabletransformation as an extrachromosomal element, or integration into thehost chromosome), the presence or absence of readily assayable orselectable markers (e.g., antibiotic resistance and metabolic markers ofone type and another), and the number of copies of the gene to bepresent in the host cell.

Vectors suitable to carry the nucleic acids of the present inventioncomprise RNA viruses, DNA viruses, lytic bacteriophages, lysogenicbacteriophages, stable bacteriophages, plasmids, viroids, and the like.The most preferred vectors are plasmids.

When preparing an expression vector the skilled artisan understands thatthere are many variables to be considered, for example, whether to use aconstitutive or inducible promoter. Inducible promoters are preferredbecause they enable high level, regulatable expression of an operablylinked gene. The skilled artisan will recognize a number of induciblepromoters that respond to a variety of inducers, for example, carbonsource, metal ions, heat, and others. The practitioner also understandsthat the amount of nucleic acid or protein to be produced dictates, inpart, the selection of the expression system. The addition of certainnucleotide sequences is useful for directing the localization of arecombinant protein. For example, a sequence encoding a signal peptidepreceding the coding region of a gene, is useful for directing theextra-cellular export of a resulting polypeptide.

Host cells harboring the nucleic acids disclosed herein are alsoprovided by the present invention. A preferred host is E. coli that hasbeen transfected or transformed with a vector that comprises a nucleicacid of the present invention.

The present invention also provides a method for constructing arecombinant host cell capable of expressing SEQ ID NO:2, or the solubleform thereof, said method comprising transforming or otherwiseintroducing into a host cell a recombinant DNA vector that comprises anisolated DNA sequence which encodes SEQ ID NO:2 or fragment thereof. Thepreferred host cell is any strain of E. coli that can accomodate highlevel expression of an exogenously introduced gene. Preferred vectorsfor expression are those which comprise SEQ ID NO:1. An especiallypreferred expression vector for use in E. coli is pPSR-23, whichcomprises nucleotide residues 202 through 807 of SEQ ID NO:1. (SeeFIGURE). Transformed host cells may be cultured under conditions wellknown to skilled artisans such that a recombinant protein is expressed,thereby producing in the recombinant host cell the MTG or MTG^(S) of theinstant invention.

For the purpose of identifying or developing new antibiotic compounds itis useful to determine compounds that bind to MTG and/or inhibit thetransglycosylase activity. The instant invention provides a screen foridentifying compounds that inhibit the enzymatic activity of MTG orMTG^(S) or fragment thereof, said screen comprising the steps of:

a) preparing and substantially purifying a recombinant MTG of theinvention; alternatively, one can start with solubilized membranes fromtransformed cells;

b) exposing said MTG to a test compound; and

c) monitoring, by any suitable means, the inhibition of enzymaticactivity of said MTG by said compound.

The MTG or MTG^(S) used in these experiments is preferably substantiallypurified as described herein. An alternative method for purifyingmembrane-bound MTG would comprise extraction from solubilized membranepreparations of cells transformed with the cloned mtg gene. Solubilizedmembranes are prepared according to well known methods.

The substrate for an MTG transglycosylase assay can be made according toart-recognized methods (See e.g. DiBerardino et al. FEBS Letters, 392,184-88 (1996). For example, the lipid precursor substrate can beprepared from Staphylococcus aureus membranes, or from the membranes ofany other suitable bacteria, UDP-Mur-Nac-pentapeptide, andUDP-N-acetyl-[¹⁴ C]glucosamine (Amersham, Buckinghamshire, UK).Transglycosylase activity is measured by the production of thepeptidoglycan polymerization product essentially by mixing the substratewith a source of MTG and monitoring the amount of [¹⁴ C]-label in thepeptidoglycan.

The screening system described above provides a means to determinecompounds that interact with the MTG of the present invention and whichmay interfere with peptidoglycan biosynthesis. This screening method maybe adapted to automated procedures such as a PANDEX® (Baxter-DadeDiagnostics) system, allowing for efficient high-volume screening forpotential inhibitory agents.

The following examples more fully describe the present invention. Thoseskilled in the art will recognize that the particular reagents,equipment, and procedures described below are merely illustrative andare not intended to limit the present invention in any manner.

EXAMPLE 1 Construction of a DNA Vector for Expressing Staphylococcusaureus mtg^(S) Gene in a Heterologous Host

Plasmid pPSR-23 (See FIGURE) is an approximately 6300 base pairexpression vector suitable for expressing a modified mtg^(S) inprocaryotic host E. coli. This plasmid contains an origin of replication(Ori), an ampicillin resistance gene (Amp), a T7 promoter, and achelating peptide and Factor Xa site in operable linkage to the codingregion of said mtg^(S) gene. The chelating peptide and Factor Xa siteare engineered onto the amino terminal end of the recombinant MTG^(S) inorder to simplify protein purification by providing a "his tag." (SeeExample 4). The parent plasmid of pPSR-23, pET16B (obtained fromNovogen, Madison, Wis.), was digested with endonucleases NdeI and BamHI.Digested pET16B was ligated to a DNA fragment bearing NdeI and BamHIsticky ends, comprising a modified mtg^(S) gene. The mtg^(S) geneligated into pPSR-23 encodes amino acid residues 68 through 269 of SEQID NO.2. The mtg^(S) gene carried on pPSR-23 is most convenientlyproduced by PCR using standard methods and oligonucleotide primerstargeted to the 5' and 3' ends of SEQ ID NO.1. The primer for synthesisat the 5' end of the gene is constructed to contain an NdeI site whilethe primer for synthesis at the 3' end of the gene is constructed tocontain a BamH1 site for cloning into pET16B.

EXAMPLE 2 Construction of a DNA Vector for Expression of mtg in aHeterologous Host

The plasmid construction method outlined in Example 1 is followed toconstruct a vector for expressing MTG comprising amino acid residues 17through 269 of SEQ ID NO.2 in a heterologous host such as E. coli.Synthesis of the mtg gene used herein is most conveniently carried outby PCR on genomic DNA from S. aureus. Synthesis is primed at the 5' endof the gene starting at nucleotide position 49 of SEQ ID NO.1 using anappropriately synthesized oligonucleotide primer. This site comprises anatural NdeI site useful for cloning into plasmid pET16B. Synthesis atthe 3' end of the mtg gene is primed using a primer constructed tocontain a BamH1 cloning site and targeted to nucleotide residuesextending through nucleotide position 807 at the 3' end of SEQ ID NO.1.

EXAMPLE 3 Expression of Staphylococcus aureus mtgS Gene in Echerichiacoli

Expression plasmid pPSR-23 was transformed into E. coli BL21(DE3)pLys5(F⁻ ompT[lon]hsdS r_(B) ⁻ m_(B) ⁻) using standard methods (Seee.g. Sambrook et al. Supra). Transformants chosen at random were testedfor the presence of pPSR-23 by agarose gel electrophoresis using quickplasmid preparations. Id. Transformants were grown overnight at 37° C.in LB medium supplemented with 100 μg/ml ampicillin. The overnightculture was diluted into fresh LB medium and allowed to grow to anO.D.₆₀₀ of 0.6 to 0.8. At that point, expression of the vector-boundmtgS gene was induced by adding 1.0 mM IPTG for a period of 4 hours. Theinduced-culture was then pelleted by centrifugation in preparation forprotein purification (See Example 4).

EXAMPLE 4 Purification of MTG^(S)

The recombinant cell pellet, isolated as described in the last step ofExample 3, was resuspended in 60 ml of 20 mM potassium phosphate, pH7.5. The cells were disrupted by passage through a French press,producing a cell extract that was centrifuged at 150,000×g for 1 hour.The MTG^(S) protein contained in the extract was purified by immobilizedmetal ion affinity chromatography (IMAC), essentially as described inU.S. Pat. No. 4,569,794, the entire contents of which is herebyincorporated by reference. Briefly, the IMAC procedure involved addingto the protein sample the following components at the indicated finalconcentrations: 0.5M NaCl, 5 mM imidazole. The sample was loaded onto aChelating Sepharose Fast Flow column (Pharmacia, 10 ml bed volume) andthe column washed twice with 35 ml each of 20 mM Tris, pH 8, 0.5 M NaCland 5 mM imidazole; 20 mM Tris, pH 8, 0.5 M NaCl and 60 mM imidazole.The bound protein was eluted from the column with 20 mM Tris, pH 8, 0.5M NaCl, 1 M imidazole.

EXAMPLE 5 Inhibition of MTG Transglycosylase Activity

Radiolabelled lipid precursor for use as substrate is prepared asdescribed in H. Hara and H. Suzuki FEBS Lett. 168, 155-60 (1984).Peptidoglycan synthesis activities are determined in 50 μl reactionscontaining 50 mM PIPES, pH 6.1, 10 mM MgCl₂, 0.2 mM DTT, 1 mM ATP, 26%DMSO, MTG or MTG^(S) sample and ¹⁴ C-labelled lipid precursor. Thereaction is incubated for 30 minutes at room temperature and filteredthrough hydrophilic Durapore PVDF membranes (0.65 μm Millipore, Bedford,Mass.). Under these conditions the synthesized peptidoglycan is retainedwhile the unincorporated labeled substrate is washed through using 0.4 Mammonium acetate in methanol. The filter-bound radioactivity isdetermined by scintillation counting.

Inhibition studies are carried out using the same reaction conditionsdescribed except that compounds to be studied for inhibitory activityare added to a final concentration between 1 mM and 10 mM.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 3                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 807 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..807                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATG AAA AGA AGC GAT AGG TAC TCA AAC TCA AA - #T GAA CAT TTT GAG CAT           48                                                                       Met Lys Arg Ser Asp Arg Tyr Ser Asn Ser As - #n Glu His Phe Glu His             1               5 - #                 10 - #                 15              - - ATG AAA CAC GAA CCT CAC TAT AAT ACG TAT TA - #T CAA CCA GTT GGC AAA           96                                                                       Met Lys His Glu Pro His Tyr Asn Thr Tyr Ty - #r Gln Pro Val Gly Lys                        20     - #             25     - #             30                  - - CCG CCG AAA AAG AAA AAA AGT AAA CGA ATA CT - #A TTA AAA ATA TTA TTA          144                                                                       Pro Pro Lys Lys Lys Lys Ser Lys Arg Ile Le - #u Leu Lys Ile Leu Leu                    35         - #         40         - #         45                      - - ACC ATT CTA ATC ATT ATC GCA TTG TTT ATT GG - #T ATC ATG TAT TTT TTA          192                                                                       Thr Ile Leu Ile Ile Ile Ala Leu Phe Ile Gl - #y Ile Met Tyr Phe Leu                50             - #     55             - #     60                          - - TCT ACA CGC GAT AAT GTG GAT GAA CTA AGA AA - #A ATT GAA AAT AAA AGT          240                                                                       Ser Thr Arg Asp Asn Val Asp Glu Leu Arg Ly - #s Ile Glu Asn Lys Ser            65                 - # 70                 - # 75                 - # 80       - - AGT TTT GTG TCA GCT GAT AAC ATG CCA GAG TA - #T GTT AAA GGT GCC TTT          288                                                                       Ser Phe Val Ser Ala Asp Asn Met Pro Glu Ty - #r Val Lys Gly Ala Phe                            85 - #                 90 - #                 95              - - ATT TCA ATG GAA GAT GAA CGA TTC TAC AAT CA - #T CAT GGA TTC GAT TTG          336                                                                       Ile Ser Met Glu Asp Glu Arg Phe Tyr Asn Hi - #s His Gly Phe Asp Leu                       100      - #           105      - #           110                  - - AAA GGT ACA ACT AGA GCT TTA TTT TCA ACG AT - #T AGC GAC AGA GAT GTG          384                                                                       Lys Gly Thr Thr Arg Ala Leu Phe Ser Thr Il - #e Ser Asp Arg Asp Val                   115          - #       120          - #       125                      - - CAA GGT GGT AGT ACC ATT ACA CAA CAA GTT GT - #C AAA AAT TAT TTT TAT          432                                                                       Gln Gly Gly Ser Thr Ile Thr Gln Gln Val Va - #l Lys Asn Tyr Phe Tyr               130              - #   135              - #   140                          - - GAT AAT GAT CGT TCA TTT ACT AGA AAA GTA AA - #A GAA TTA TTT GTA GCT          480                                                                       Asp Asn Asp Arg Ser Phe Thr Arg Lys Val Ly - #s Glu Leu Phe Val Ala           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - CAT CGA GTT GAA AAA CAA TAT AAT AAG AAC GA - #A ATT TTA AGC TTT        TAT      528                                                                    His Arg Val Glu Lys Gln Tyr Asn Lys Asn Gl - #u Ile Leu Ser Phe Tyr                          165  - #               170  - #               175              - - TTA AAT AAT ATT TAC TTT GGG GAT AAT CAA TA - #T ACG CTT GAG GGC GCA          576                                                                       Leu Asn Asn Ile Tyr Phe Gly Asp Asn Gln Ty - #r Thr Leu Glu Gly Ala                       180      - #           185      - #           190                  - - GCA AAC CAT TAC TTT GGA ACA ACC GTG AAT AA - #A AAT AGT ACA ACA ATG          624                                                                       Ala Asn His Tyr Phe Gly Thr Thr Val Asn Ly - #s Asn Ser Thr Thr Met                   195          - #       200          - #       205                      - - TCT CAC ATA ACA GTT TTA CAA AGC GCT ATT TT - #A GCT AGT AAA GTC AAT          672                                                                       Ser His Ile Thr Val Leu Gln Ser Ala Ile Le - #u Ala Ser Lys Val Asn               210              - #   215              - #   220                          - - GCA CCT AGC GTA TAT AAT ATC AAT AAT ATG TC - #A GAG AAT TTT ACG CAA          720                                                                       Ala Pro Ser Val Tyr Asn Ile Asn Asn Met Se - #r Glu Asn Phe Thr Gln           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - CGT GTA AGC ACG AAC TTA GAA AAA ATG AAG CA - #A CAA AAT TAT ATC        AAT      768                                                                    Arg Val Ser Thr Asn Leu Glu Lys Met Lys Gl - #n Gln Asn Tyr Ile Asn                          245  - #               250  - #               255              - - GAA ACA CAA TAT CAA CAG GCT ATG TCA CAA TT - #A AAT CGT                  - #    807                                                                    Glu Thr Gln Tyr Gln Gln Ala Met Ser Gln Le - #u Asn Arg                                   260      - #           265                                         - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 269 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Lys Arg Ser Asp Arg Tyr Ser Asn Ser As - #n Glu His Phe Glu His        1               5 - #                 10 - #                 15              - - Met Lys His Glu Pro His Tyr Asn Thr Tyr Ty - #r Gln Pro Val Gly Lys                   20     - #             25     - #             30                  - - Pro Pro Lys Lys Lys Lys Ser Lys Arg Ile Le - #u Leu Lys Ile Leu Leu               35         - #         40         - #         45                      - - Thr Ile Leu Ile Ile Ile Ala Leu Phe Ile Gl - #y Ile Met Tyr Phe Leu           50             - #     55             - #     60                          - - Ser Thr Arg Asp Asn Val Asp Glu Leu Arg Ly - #s Ile Glu Asn Lys Ser       65                 - # 70                 - # 75                 - # 80       - - Ser Phe Val Ser Ala Asp Asn Met Pro Glu Ty - #r Val Lys Gly Ala Phe                       85 - #                 90 - #                 95              - - Ile Ser Met Glu Asp Glu Arg Phe Tyr Asn Hi - #s His Gly Phe Asp Leu                  100      - #           105      - #           110                  - - Lys Gly Thr Thr Arg Ala Leu Phe Ser Thr Il - #e Ser Asp Arg Asp Val              115          - #       120          - #       125                      - - Gln Gly Gly Ser Thr Ile Thr Gln Gln Val Va - #l Lys Asn Tyr Phe Tyr          130              - #   135              - #   140                          - - Asp Asn Asp Arg Ser Phe Thr Arg Lys Val Ly - #s Glu Leu Phe Val Ala      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - His Arg Val Glu Lys Gln Tyr Asn Lys Asn Gl - #u Ile Leu Ser Phe        Tyr                                                                                             165  - #               170  - #               175             - - Leu Asn Asn Ile Tyr Phe Gly Asp Asn Gln Ty - #r Thr Leu Glu Gly Ala                  180      - #           185      - #           190                  - - Ala Asn His Tyr Phe Gly Thr Thr Val Asn Ly - #s Asn Ser Thr Thr Met              195          - #       200          - #       205                      - - Ser His Ile Thr Val Leu Gln Ser Ala Ile Le - #u Ala Ser Lys Val Asn          210              - #   215              - #   220                          - - Ala Pro Ser Val Tyr Asn Ile Asn Asn Met Se - #r Glu Asn Phe Thr Gln      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Arg Val Ser Thr Asn Leu Glu Lys Met Lys Gl - #n Gln Asn Tyr Ile        Asn                                                                                             245  - #               250  - #               255             - - Glu Thr Gln Tyr Gln Gln Ala Met Ser Gln Le - #u Asn Arg                              260      - #           265                                         - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 807 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: mRNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - AUGAAAAGAA GCGAUAGGUA CUCAAACUCA AAUGAACAUU UUGAGCAUAU GA -             #AACACGAA     60                                                                 - - CCUCACUAUA AUACGUAUUA UCAACCAGUU GGCAAACCGC CGAAAAAGAA AA -            #AAAGUAAA    120                                                                 - - CGAAUACUAU UAAAAAUAUU AUUAACCAUU CUAAUCAUUA UCGCAUUGUU UA -            #UUGGUAUC    180                                                                 - - AUGUAUUUUU UAUCUACACG CGAUAAUGUG GAUGAACUAA GAAAAAUUGA AA -            #AUAAAAGU    240                                                                 - - AGUUUUGUGU CAGCUGAUAA CAUGCCAGAG UAUGUUAAAG GUGCCUUUAU UU -            #CAAUGGAA    300                                                                 - - GAUGAACGAU UCUACAAUCA UCAUGGAUUC GAUUUGAAAG GUACAACUAG AG -            #CUUUAUUU    360                                                                 - - UCAACGAUUA GCGACAGAGA UGUGCAAGGU GGUAGUACCA UUACACAACA AG -            #UUGUCAAA    420                                                                 - - AAUUAUUUUU AUGAUAAUGA UCGUUCAUUU ACUAGAAAAG UAAAAGAAUU AU -            #UUGUAGCU    480                                                                 - - CAUCGAGUUG AAAAACAAUA UAAUAAGAAC GAAAUUUUAA GCUUUUAUUU AA -            #AUAAUAUU    540                                                                 - - UACUUUGGGG AUAAUCAAUA UACGCUUGAG GGCGCAGCAA ACCAUUACUU UG -            #GAACAACC    600                                                                 - - GUGAAUAAAA AUAGUACAAC AAUGUCUCAC AUAACAGUUU UACAAAGCGC UA -            #UUUUAGCU    660                                                                 - - AGUAAAGUCA AUGCACCUAG CGUAUAUAAU AUCAAUAAUA UGUCAGAGAA UU -            #UUACGCAA    720                                                                 - - CGUGUAAGCA CGAACUUAGA AAAAAUGAAG CAACAAAAUU AUAUCAAUGA AA -            #CACAAUAU    780                                                                 - - CAACAGGCUA UGUCACAAUU AAAUCGU          - #                  - #                807                                                                   __________________________________________________________________________

We claim:
 1. An isolated monofunctional glycosyltransferase fromStaphylococcus aureus having the amino acid sequence:

    Met Lys Arg Ser Asp Arg Tyr Ser Asn Ser                                          1               5                 10                                         - Asn Glu His Phe Glu His Met Lys His Glu                                                     15                  20                                        - Pro His Tyr Asn Thr Tyr Tyr Gln Pro Val                                                     25                  30                                        - Gly Lys Pro Pro Lys Lys Lys Lys Ser Lys                                                     35                  40                                        - Arg Ile Leu Leu Lys Ile Leu Leu Thr Ile                                                     45                  50                                        - Leu Ile Ile Ile Ala Leu Phe Ile Gly Ile                                                     55                  60                                        - Met Tyr Phe Leu Ser Thr Arg Asp Asn Val                                                     65                  70                                        - Asp Glu Leu Arg Lys Ile Glu Asn Lys Ser                                                     75                  80                                        - Ser Phe Val Ser Ala Asp Asn Met Pro Glu                                                     85                  90                                        - Tyr Val Lys Gly Ala Phe Ile Ser Met Glu                                                     95                 100                                        - Asp Glu Arg Phe Tyr Asn His His Gly Phe                                                    105                 110                                        - Asp Leu Lys Gly Thr Thr Arg Ala Leu Phe                                                    115                 120                                        - Ser Thr Ile Ser Asp Arg Asp Val Gln Gly                                                    125                 130                                        - Gly Ser Thr Ile Thr Gln Gln Val Val Lys                                                    135                 140                                        - Asn Tyr Phe Tyr Asp Asn Asp Arg Ser Phe                                                    145                 150                                        - Thr Arg Lys Val Lys Glu Leu Phe Val Ala                                                    155                 160                                        - His Arg Val Glu Lys Gln Tyr Asn Lys Asn                                                    165                 170                                        - Glu Ile Leu Ser Phe Tyr Leu Asn Asn Ile                                                    175                 180                                        - Tyr Phe Gly Asp Asn Gln Tyr Thr Leu Glu                                                    185                 190                                        - Gly Ala Ala Asn His Tyr Phe Gly Thr Thr                                                    195                 200                                        - Val Asn Lys Asn Ser Thr Thr Met Ser His                                                    205                 210                                        - Ile Thr Val Leu Gln Ser Ala Ile Leu Ala                                                    215                 220                                        - Ser Lys Val Asn Ala Pro Ser Val Tyr Asn                                                    225                 230                                        - Ile Asn Asn Met Ser Glu Asn Phe Thr Gln                                                    235                 240                                        - Arg Val Ser Thr Asn Leu Glu Lys Met Lys                                                    245                 250                                        - Gln Gln Asn Tyr Ile Asn Glu Thr Gln Tyr                                                    255                 260                                        - Gln Gln Ala Met Ser Gln Leu Asn Arg                                                        265                                                      

which is SEQ ID NO
 2. 2. An isolated soluble form of monofunctionalglycosyltransferase from Staphylococcus aureus having the amino acidsequence which is defined by amino acid residues 68 through 269 of SEQID NO.2.
 3. An isolated form of monofunctional glycosyltransferase fromStaphylococcus aureus having the amino acid sequence which is defined byamino acid residues 17 through 269 of SEQ ID NO.
 2. 4. An isolatedmonofunctional glycosyltransferase from Staphylococcus aureus consistingessentially of the amino acid sequence shown in SEQ ID NO.
 2. 5. Anisolated soluble form of monofunctional glycosyltransferase fromStaphylococcus aureus consisting essentially of amino acid residues 68through 269 of SEQ ID NO.
 2. 6. An isolated form of monofunctionalglycosyltransferase from Staphylococcus aureus consisting essentially ofamino acid residues 17 through 269 of SEQ ID NO.
 2. 7. A method foridentifying a compound that inhibits the transglycosylase activity of aStaphylococcus aureus monofunctional glycosyltransferase, comprising thefollowing steps:a) admixing:i) a protein selected from the groupconsisting of substantially pure Staphylococcus aureus monofunctionalglycosyltransferase and a fragment thereof that retains transglycosylaseactivity, ii) a suitable substrate for Staphylococcus aureusmonofunctional glycosyltransferase, and iii) a test compound to bestudied for inhibitory activity against said transglycosylase activity;b) incubating said protein, said substrate, and said test compoundtogether; c) measuring transglycosylase activity by any suitable means;and d) comparing transglycosylase activity of step c) totransglycosylase activity observed upon incubating said protein and saidsubstrate as in step b) in the absence of said test compound.
 8. Themethod of claim 7, wherein said fragment that retains transglycosylaseactivity is selected from the group consisting of a form ofStaphylococcus aureus monofunctional glycosyltransferase comprisingamino acid residues 17 through 269 of SEQ ID NO.:2, and a soluble formof Staphylococcus aureus monofunctional glycosyltransferase.
 9. Themethod of claim 8, wherein said soluble form of Staphylococcus aureusmonofunctional glycosyltransferase comprises amino acid residues 68through 269 of SEQ ID NO:2.
 10. The method of claim 7, wherein saidsubstrate of step a)ii) comprises a radiolabelled lipid precursor ofpeptidoglycan polymerization product, formation of which is catalyzed byStaphylococcus aureus monofunctional glycosyltransferase.
 11. The methodof claim 7, wherein said substrate of step a)ii) comprises a lipidprecursor of peptidoglycan polymerization product, formation of which iscatalyzed by Staphylococcus aureus monofunctional glycosyltransferase,UDP-Mur-Nac-pentapeptide, and UDP-N-acetyl-[¹⁴ C]glucosamine.
 12. Themethod of claim 7, wherein said incubating of step b) is carried out in50 mM piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.1; 10 mM MgCl₂ ;0.2 mM dithiothreitol; 1 mM adenosine triphosphate; and 26%dimethylsulfoxide.
 13. The method of claim 7, wherein said test compoundis present at a final concentration between 1 mM and 10 mM.